Mitochondria from patients with Kearns-Sayre syndrome harboring large-scale rearrangements of human mitochondrial DNA (mtDNA; both partial deletions and a partial duplication) were introduced into human cells lacking endogenous mtDNA. Cytoplasmic hybrids containing 100% wild-type mtDNA, 100% mtDNA with partial duplications, and 100% mtDNA with partial deletions were isolated and characterized. The cell lines with 100% deleted mtDNAs exhibited a complete impairment of respiratory chain function and oxidative phosphorylation. In contrast, there were no detectable respiratory chain or protein synthesis defects in the cell lines with 100% duplicated mtDNAs. Unexpectedly, the mass of mtDNA was identical in all cell lines, despite the fact that different lines contained mtDNAs of vastly different sizes and with different numbers of replication origins, suggesting that mtDNA copy number may be regulated by tightly controlled mitochondrial dNTP pools. In addition, quantitation of mtDNA-encoded RNAs and polypeptides in these lines provided evidence that mtDNA gene copy number affects gene expression, which, in turn, is regulated at both the post-transcriptional and translational levels.

A T 3 G mutation at position 8993 in human mitochondrial DNA is associated with the syndrome neuropathy, ataxia, and retinitis pigmentosa and with a maternally inherited form of Leigh's syndrome. The mutation substitutes an arginine for a leucine at amino acid position 156 in ATPase 6, a component of the F 0 portion of the mitochondrial ATP synthase complex. Fibroblasts harboring high levels of the T8993G mutation have decreased ATP synthesis activity, but do not display any growth defect under standard culture conditions. Combining the notions that cells with respiratory chain defects grow poorly in medium containing galactose as the major carbon source, and that resistance to oligomycin, a mitochondrial inhibitor, is associated with mutations in the ATPase 6 gene in the same transmembrane domain where the T8993G amino acid substitution is located, we created selective culture conditions using galactose and oligomycin that elicited a pathological phenotype in T8993G cells and that allowed for the rapid selection of wild-type over T8993G mutant cells. We then generated cytoplasmic hybrid clones containing heteroplasmic levels of the T8993G mutation, and showed that selection in galactose-oligomycin caused a significant increase in the fraction of wild-type molecules (from 16 to 28%) in these cells.A T 3 G transversion at human mtDNA 1 position 8993 (1) is associated with a multisystemic syndrome characterized clinically by neuropathy, ataxia, and retinitis pigmentosa (NARP) (2), and with a maternally inherited form of Leigh's syndrome (MILS) (3, 4), a fatal encephalopathy of infancy. The mutation results in the conversion of leucine to arginine at amino acid 156 in ATPase 6, one of the two mtDNAencoded subunits of the F 0 portion of the mitochondrial ATPase complex (complex V). It has been reported that very high levels of mutant mtDNA are required for the clinical phenotype to be expressed (3), suggesting that this mutation is "recessive" in nature.Cultured cells harboring high levels (Ͼ95%) of the T8993G mutation have decreased ATP synthase activity (5, 6), implying that a defect in oxidative ATP production is the most likely cause of both disorders. Nevertheless, cultured fibroblasts from NARP and MILS patients do not display any growth abnormalities under standard culture conditions, despite often being homoplasmic for the mutation (i.e. containing 100% mutant mtDNA). Nevertheless, we hypothesized that we could identify culture conditions that would not only allow for selection against mutant cells, but would also affect the intracellular proportion of mutant mtDNAs.We took advantage of two factors that eventually allowed us to develop such a system. First, fibroblasts from patients with diverse mitochondrial disorders grow poorly when glucose is replaced by galactose as the sole carbon source (7). Second, resistance to the antibiotic oligomycin, an inhibitor of mitochondrial function, is associated with at least two mutations in the yeast ATPase 6 gene (8) and with at least one mutation in the hamster ATPas...